Infection of CD4-positive lymphocytes by Human Immunodeficiency Virus (HIV-1) involves a complex pathway. This is also true for infection by other retroviruses. To chart the course of HIV-1 infection, as an example, it is absolutely necessary to have a sensitive biologically relevant infectivity assay that determines primary infection. Such an assay is also critical for the valid evaluation of neutralization or inhibition by antibodies or antiviral agents. Infection of target cells by HIV-1, for example, can be detected by several methods, including observation of virus induced cytopathic effects, such as syncytia formation; immunofluorescence assays to detect expression of vital antigens; antigen capture assays that detect the release of viral proteins, such as p24; and detection of reverse transcriptase activity in the supernatants of infected cells. Unfortunately, although these assays are sensitive for the detection of infection of neoplastic T-cell lines, they are of little use when peripheral blood mononuclear cells (PBMC) are used as the target cells, particularly when the assays are employed at a very early time. This is because, typically, a very low percentage of the cells are infected under in-vitro conditions by primary HIV-1 isolates. Infection must be allowed to progress for an extended period of time (usually a minimum of six days) before it can be detected by conventional methods. Further, many HIV-1 isolates infect PBMC without inducing syncytia formation. Thus, the applicability of these conventional assays for the detection of infection by HIV-1 is somewhat limited.
The major objective of the present invention was the development of a sensitive infection assay, which would allow for the accurate study of the human retrovirus infection process and would also permit valid evaluation of potentially therapeutic antiviral agents. Therefore, we tried the molecular amplification technique known as NASBA (Kievits et al., 1991), which has proven to be a particularly effective and sensitive means of amplification for detecting the RNA genome of HIV-1.
NASBA is an isothermal method of specific amplification that relies on a set of two primers specific for the target sequence. The antisense primer (P1) has a 5' overhang that encodes the T7 RNA polymerase promoter; the sense primer (P2) is specific for a region upstream of the P1 annealing site. During the early stages of the reaction, a cDNA copy of the target area is generated through the coordinated activities of reverse transcriptase and RNase H. The resulting cDNA encodes a functional T7 RNA polymerase promoter at one end. Amplification can therefore occur through the action of the T7 RNA polymerase. The final NASBA product consists of single stranded copies of antisense RNA representing the area targeted by the primers.
We have found NASBA to be useful as an endpoint assay for the detection of infection of PBMC and CEM50 (CEM) cells by HIV-1. We used the assay to detect both HIV-1 genomic RNA and spliced HIV-1 mRNA transcript target sequences resulting from primary infection, with little contribution from secondary infection.